The pyrimidine deoxyribonucleoside thymidine is useful as a pharmaceutical intermediate. It is particular important for the chemical synthesis of the AZT, also known as zidovidine or azidothymidine. This is an antiretroviral drug of the nucleoside reverse transcriptase inhibitor (NRTI) class, which, under the trademark Retrovir, was the first drug licensed to treat HIV infection.
HIV/AIDS is usually treated with combinations of three or more antiretroviral drugs (De Clercq, Med. Chem. Res. 13: 439-478, 2004). AZT has an important and expanded role as a major component of combination therapies and is sold under a variety of trademarks including Retrovir, Zidovir, Viro-Z, Aviro-Z and Zido-H.
AZT is particularly valuable when combined with the NRTI drug 3TC (lamivudine). These two drugs are available, formulated in a single pill, under the trademarks Combivir and Duovir. A triple NRTI combination of AZT, 3TC and abacavir is sold under the trademark Trizivir. Other NRTI drugs used in conjunction with AZT include didanosine, emtricitabine and zalcitabine.
AZT is also useful in combination with HIV protease inhibitor drugs including amprenavir, atazanavir, indinavir, ritonavir and saquinavir, and with non-nucleoside reverse transcriptase inhibitors (NNRTI) including delavirdine, efavirenz and nevirapine.
AZT is the only anti-HIV drug approved for use during pregnancy (Lyall, et al., HIV Med. 2: 314-334, 2001). In 1997 about 600,000 children died of AIDS contracted by mother to baby transmission. AZT, taken in the last trimester of pregnancy, may reduce the risk of viral transmission by 67% (Mitchla & Sharland, Expert Opin. Pharmacother. 1: 239-248, 2000).
Fermentation technology is an attractive alternative to chemical synthesis for commercial preparation of the large quantities of thymidine required for AZT manufacture. Fermentation processes are well established in industry as a means to produce biological molecules such as antibiotics, amino acids and vitamins at large scale and relatively low cost (Atkinson, & Mavituna, Biochemical Engineering and Biotechnology Handbook, 2nd Edition, New York, Stockton Press, 1991).
However thymidine is not usually found in its “free” form in nature, but produced as the monophosphate thymidylic acid and incorporated into DNA as the triphosphate. Biological systems do not naturally produce significant amounts of thymidine, hence a mutant or engineered organism is required.
In EP 0,344,937 there are disclosed strains of Brevibacterium selected to produce thymidine on aerobic cultivation. Reference is also made to a Japanese patent publication No. 39-16345, which teaches the use of a mutant strain of Bacillus subtlis in the fermentative production of a polysaccharide containing thymidine.
In U.S. Pat. No. 5,213,972 (McCandliss & Anderson), the entire contents of which are incorporated herein by reference and to which the reader is specifically referred, a process is disclosed for the production of pyrimidine deoxyribonucleosides (PdN) such as thymidine and 2′-deoxyuridine. A replicatable microorganism is taught, incorporating and expressing a DNA sequence encoding a PdN phosphohydrolase that converts a PdN monophosphate to a PdN.
More particularly, McCandliss & Anderson, supra, describe a fermentation method that can be used to produce thymidine that involves the expression of deoxythymidylate phosphohydrolase (dTMPase) from the Bacillus subtlis bacteriophage PBS1. This type of enzyme is found in nature expressed by bacteriophages that incorporate PdNs such as 2′-deoxyuridine or 5-hydroxymethyl-2′-deoxyuridine in their DNA in place of thymidine.
In the thymidine fermentation described in U.S. Pat. No. 5,213,972 the enzymes that degrade thymidine, (thymidine phosphorylase and uridine phosphorylase), have been removed by mutation so that thymidine accumulates. Thus, the use of the dTMPase enzyme helps create the pathway to allow thymidine synthesis. An expression of dTMPase alone, however, may not assure a commercially viable level of thymidine production
In WO 01/02580, the entire contents of which are also incorporated herein by reference and to which the reader is also specifically referred, there are described improvements which result in the enhanced production of thymidine by cells expressing dTMPase.
However a problem in the manufacture of biologically produced thymidine lies in the concomitant production of 2′-deoxyuridine (UdR) in the fermentation process. The two molecules have similar properties, differing in structure by a single methyl group, and separation during downstream processing is difficult and expensive. For a pharmaceutical application, such as the synthesis of AZT, high purity thymidine may be required with low levels of UdR.
It would be beneficial if thymidine biologically produced by fermentation gave significantly reduced levels of UdR compared to current processes, such that the requirement for downstream purification to remove this material were minimised or eliminated.
By significantly reduced is meant that (contaminating) levels of UdR constitute less than 25%, more preferably still less than 10%, more preferably still less than 5% through 4%, 3%, and 2%, to levels of 1% and less in the fermented thymidine product.
Ideally, “high” levels of thymidine should be attained. Levels of thymidine can be given as “specific productivity” figures where the measure is determined 4-6 hours after induction. Levels of greater than 5 mg TdR/1/hr/g dry cell weight, more particularly greater than 10 mg TdR/1/hr/g dry cell weight and more preferably specific productivity figures of greater than 15 through 20 and 25 are preferred.
These levels should most preferably be obtained with the reduced levels of UdR described above and thus the figures can be combined such that, for example a TdR titre of 5 g/l might be obtained containing less than, for example 5% UdR.
Indeed the prior art process, using the plasmid pCG532 in host strain CMG2451, as disclosed in WO 01/02580, produces an average UdR content in shake flask tests of 34.5%. At such high levels downstream processing to separate thymidine from UdR is difficult and costly.
It is an aim of the present invention to modify thymidine producing organisms such that they produce thymidine without significant concomitant production of UdR.
As used herein the term “organism” includes an organism which can produce thymidine through expression of DNA encoded in its chromosome (as disclosed in, for example, EP 0344937) or DNA which it hosts (as disclosed in, for example, U.S. Pat. No. 5,213,972 and WO 01/02580). This host DNA may be present as one or more DNA constructs, such as, for example, one or more plasmids.
In particular it is an aim to provide an improvement in the quality of thymidine produced as compared to the construct/host combination disclosed in WO 01/02580.
It is a further aim to ensure that any gene modification made to a construct is stable such that when the construct is introduced into a host cell, and the combination grown, the host is unable to propagate itself without the construct being present. i.e. if the construct is lost, the organism dies.